Antigen selection and affinity maturation of autoantibodies leave a genetic imprint indicative of the type of antigen that is bound in vivo. Therefore, a detailed investigation of genetic mechanisms that generate the immunological diversity of autoantibodies should provide crucial information about the induction of autoimmunity. The principle of antigen selection has been tested with murine anti-DNA antibodies and recent studies suggest that it also applies to a wide range of autoantibodies that arise in human autoimmune disease. This application proposes to analyze by in vitro mutagenesis three autospecificities that are typical of Systemic Lupus Erythematosus: anti- double stranded DNA, anti-histone, and anti-snRNP. In particular, the effects of V gene use, rearrangement, and somatic mutation will be evaluated in three antibodies that have dual specificity and bind either histones or snRNP in addition to DNA. The focus of the studies will be to learn: a) the relative contributions of the different diversity-generating mechanisms, and b) the order in which the three specificities arise. It is hoped that conclusions from these studies will be relevant for the etiology and possible treatment of Systemic Lupus Erythematosus. The experiments described have the following aims: Specific Aim 1: Clone and express anti-DNA V genes. The heavy (H) and light (L) chain V genes used by three hybridomas 3H9, LG8-1, and 1-42 will be cloned and manipulated in vitro, in order to determine the structural basis for dsDNA, nucleosome, and SmD binding. These experiments will involve chain recombination, VHCDR3 grafting, and site-directed mutagenesis. Specific Aim 2: Test antibody specificity for dsDNA conformation. Purified mutant anti-dsDNA antibodies will be used to establish whether DNA curvature improves DNA binding or not. If conformational preference is found, then: a) The size and location of bound DNA sequences will be established using Exonuclease III and ethylation interference footprinting. b) Single chain antibody fragments (scFv) will be constructed to distinguish whether preference is intrinsic to each combining site or is a consequence of IgG structure. Specific Aim 3: Explore antibody specificity for dsDNA sequence. In order to evaluate the potential for DNA sequence specificity, oligonucleotides of initially random composition will be processed through several cycles of affinity enrichment and PCR purification. Affinity chromatography will be on anti-dsDNA antibodies or anti-dsDNA scFv linked to functional domains from the transcriptional factor GCN4 and from protein A. Substrates containing a palindromic core sequence will be used to reduce the lateral displacement of bound antibodies.